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31 Cards in this Set
- Front
- Back
what is FLOURESCENCE?
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Absorption of one wavelngth, emission of a longer wavelngth with lower energy.
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what 3 steps occur in flourescence?
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1. Excitation with UV lite
2. Reorientation 3. Emission of Visible lite |
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what 3 types of molecules flouresce the best?
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1. Molecules w/ conjugated double bonds.
2. Molecules w/ ring closures 3. Metal chelated molecules. |
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What is Quantum efficiency?
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the YIELD:
Ratio of #flouresc molecules to #excited molecules. |
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how many components are in a flourometer, and what are they?
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6:
1. Stable power supply 2. Light source 3. Primary filter 4. Sample holder 5. Secondary filter 6. Detector |
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what does power get supplied TO?
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-Uv light source
-Photodetector. |
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What type of light source is in the flourometer?
-What type of bulbs can be used? |
-UV light emitting at 200-800 nanometers.
-Hydrogen, Deuterium, Xenon/Mercury Arc |
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How many filters?
What are they? |
Two.
-Primary filter -Secondary filter -Both are Wratten (gelatinous) |
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What does each filter do?
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1ary selects the absorption wavelength.
2ary selects the flourescent wavelength, has wide bandpass. |
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what are the limitations for the sample holder in flourometers?
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-cuvettes made of
-GLASS if excit wvlngth is over 320 nanometers -QUARTZ if less. or just use POLYSTYRENE |
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what is the detector in a flourometer?
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PMT, which feeds the meter readout with info.
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what angle is between the two filters?
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90 degrees to prevent overlap of flourescent and excitation wavelengts.
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what 3 types of instruments use flourometry in them?
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1. Fluorometers
2. Spectrofluorometers 3. Specific use instruments. |
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whats the difference between a flourometer and spectrofluor.?
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-fluormtr uses a FILTER, and Mercury arc noncontin. lamp.
-Spectrofl. uses a GRATING, and Xenon arc continuous output lamp. -Flour has to chng filters, spectro scans over various wavelengths to select. |
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how do they operate differently?
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Flourometer has to chng filters,
Spectro scans over various wavelengths to select. |
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what is each instrument used in?
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Flourometer: clinical practice
Spectrofluorometer: research b/c it is more sophisticated. |
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what are 2 types of specfc use flourometers?
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1. Morphine analyzer
2. Calcium titrator |
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what is the 2 ADVANTAGES of flourometry?
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1. Sensitivity
2. Specificity |
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how sensitive is flourometry?
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1000x more than absorption photometry!
Detection of 0.1 ppBILLION Absorpn detects 0.1 ppMILLION |
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how many problems are associated w/ flourometry?
What are they? |
A LOT! - 6:
1. Limited choice of standard. 2. Quenching 3. Photodecomposition 4. pH effect 5. Temp effect 6. Other contamination |
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why is the reference compound or standard limited?
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because the analyte being measured may not be available - morphine for e.g.
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What is quenching?
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Returning to ground state by a mechanism other than emission, results are decreased
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What 4 things cause quenching?
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1. Molecular dissociation
2. Interaction w/ other stuff 3. Solution contaminants 4. Sample concentration |
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why would molecules dissociate?
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if the excitation wavelength has more energy than the bond strength of samples
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what solution contaminants cause quenching?
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Halides
oxygen, nitrogen compounds. NaCL so can't use saline diluent. |
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how does sample concentration affect flourescence?
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If the sample is too concentrated, all the light is absorbed by molecules at one side of cuvette; must dilute to correct.
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What is "Photodecomposition"?
How does it affect results? How is it corrected? |
the decomposition of sample due to intense UV lite.
Results are decreased. Lower intensity or complex w/ substance to alter absorption peak. |
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How does pH affect flouresc.?
How does it affect results? How is it corrected? |
By modifying the resonance structure of a molecule.
Results will vary. Control the pH to correct. |
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How does Temp effect flouresc?
How does it affect results? How is it corrected? |
by promoting vibration and collision, thus energy loss.
Results: decreased Correct: maintain T or cool soln |
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What can contaminate sample?
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Stopcock grease
Cleaning agents Chemical impurities Filter paper residues. |
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What are 2 clinical applications of flourescence?
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1. Meausring trace quantities of stuff: Drugs (monitor salicylate, heart therapy), Enzymes, Amino acids.
2. Immunoflouresc. w/ Ab tag |